PROJECT SUMMARY The Gram-negative anaerobe Fusobacterium nucleatum is a key colonizer in the development of oral biofilms, or dental plaque, and also known for its association with human diseases including oral infections, adverse pregnancy outcomes, and colorectal cancer. F. nucleatum has an inherent ability to interact or aggregate with many early and late colonizers of the oral biofilms. It induces inflammatory responses and preterm birth in rodent models of infection, as well as promoting colorectal carcinogenesis in vivo. Despite its pathogenic potential, we have limited knowledge about the mechanisms of fusobacterial virulence and associated factors; to date only six fusobacterial factors have been reported, i.e. FomA, FadA, Fap2, RadD, aid1, and FAD-I, although more than 2,000 open reading frames are annotated in the genome of many F. nucleatum strains. A major obstacle limiting progress is the lack of robust genetic tools and systematic investigations. We have begun to tackle this problem, successfully developing a facile gene deletion system for F. nucleatum, and generating a large library of random transposon mutants with 10-fold genome coverage. With these previously unavailable tools, we aim to identify virulence determinants of F. nucleatum using multiple complementary approaches including forward and reverse genetics, cryo-electron tomography, biochemical methods, and rodent models of infection. By electron microscopy, we discovered that F. nucleatum produces outer membrane tubules (OMTs) and identified a key factor required for OMT formation. By gene deletion, we found pathways that mediate oxidative stress defense and host cell adherence and invasion. By transposon mutagenesis, we revealed several biofilm-associated factors that are required for production of hydrogen sulfide, which largely contributes to bad breath, or halitosis. In this application, we aim to elucidate the mechanism of OMT biogenesis and OMT-mediated pathogenesis, reveal the mechanisms of host immune defense and host cell interactions, and establish the role of biofilm- associated factors in halitosis, polymicrobial interactions, and bacterial virulence. Our studies will greatly contribute to our understanding of the molecular virulence mechanisms of this pathogen and significantly move the field forward with the application of newly developed genetic tools and advanced technology. The results generated from these studies may also provide promising targets for the future development of therapeutic strategies.